Molecular analysis for gene abnormalities in congenital multiple anomalies and prenatal DNA diagnosis.

先天性多发性异常基因异常的分子分析和产前DNA诊断。

基本信息

批准号：
02807156
负责人：
IWASAKI Katsuhiko
金额：
$ 1.09万
依托单位：
Tokai University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02807156/
关键词：
PCNA DNA polymerase congenital anomalies VNTR PCNA DNAポリメラ-ゼβ γークリスタリン DNA複製遺伝子

项目摘要

High molecular weight DNAS were purified from the placentas or autopsied tissues of severe congenital malformations. Fifteen DNA samples were examined for the abnormal restriction fragments by Southern blot analysis using cDNAs for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-delta auxiliary protein(PCNA)as probes. The DNA samples were digested with 10 restriction endonucleases. In one case of congenital malformations, the 3 probes detected abnormal restriction fragments in the DNA digests of 3-5 restriction enzymes, respectively. All abnormal bands were seen heterozygously. Clinical examination of the patient(MY)revealed microcephalia, urogenital anomaly, arthrogryposis and scoliosis. In the other 14 DNA samples, no abnormal bands were detected.DNA polymerase delta auxiliary protein(PCNA)is an essential element for DNA replication in eukaryotes and the gene is highly conserved throughout the plant and animal kingdoms(Suzuka et al. PNAS 1989). Yamaguchi et al. (MCB 1990)demonstrated that Drosophila HOX proteins specifically bind to the 5'-flanking region of Drosophila PCNA gene. In the same region of the human PCNA gene, a perfect octamer motif ATTTGCAT is found. These observations strongly suggest that the expression of PCNA is under control of the HOX proteins. Therefore, one of the regulatory pathways necessary for programmed morphogenesis would be described hierarchically as follows : expression of the homeobox genes-- control of the DNA replication genes-- regulation of the cell proliferation-- normal development.On the assamption that variable number of tandem repeat(VNTR)sequence may encode hotspots for recombinational activity and be involved in the genesis of polygene abnormality, we used VNTR marker(pYNH24)for the screening of polygene abnormality in congenital malformations. The PYNH24 probe successfully detected abnormal bands in the DNA sample from case MY.

从胎盘或严重先天性畸形的尸体组织中纯化高分子量DNA。通过使用cDNA进行DNA聚合酶-Alpha，DNA聚合酶-Beta和DNA聚合酶 - 二硫代酶辅助蛋白（PCNA）作为探针，检查了15个DNA样品的异常限制片段。用10个限制性核酸内切酶消化DNA样品。在一种先天性畸形的情况下，这3个探针分别检测到3-5个限制性酶的DNA消化中的异常限制片段。所有异常带均无杂合。对患者（MY）的临床检查表明，泌尿生殖器异常，关节炎和脊柱侧弯。在其他14个DNA样品中，未检测到异常带。DNA聚合酶三角辅助蛋白（PCNA）是真核生物中DNA复制的重要元素，并且该基因在整个动植物王国中高度保守（Suzuka etal。PNAS1989）。 Yamaguchi等。（MCB 1990）证明果蝇HOX蛋白特异性结合了果蝇PCNA基因的5'-频型区域。在人PCNA基因的同一区域中，发现了一个完美的八聚体基序。这些观察结果强烈表明PCNA的表达在HOX蛋白的控制下。因此，在层次上描述了编程形态发生所需的调节途径之一，如下所示：同型基因基因的表达 - 对DNA复制基因的控制 - 细胞增殖的调节 - 正常开发 - 正常开发。通过诱导序列的可变数量（VNTR）序列可变化，可以用来重新构造和重新构造，我们在重新构造中涉及综合范围。标记物（Pynh24）用于筛选先天性畸形中的多边形异常。 PYNH24探针从案例MY中成功检测到DNA样品中的异常带。